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On-beads digestion of streptavidin-bound proteins

EB Erica M. Briggs
PM Paolo Mita
XS Xiaoji Sun
SH Susan Ha
NV Nikita Vasilyev
ZL Zev R. Leopold
EN Evgeny Nudler
JB Jef D. Boeke
SL Susan K. Logan
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Streptavidin beads were washed twice with 1 mL 50 mM NH4HCO3 to exchange the buffer. Washed beads were then resuspended in 50 μl 50 mM NH4HCO3 containing 20 ng/μl trypsin/Lys-C (Promega) followed by overnight incubation at 37 °C with vigorous mixing in a thermoshaker (Eppendorf). After incubation beads were pelleted and supernatants were transferred to new tubes. Samples were acidified by adding 5 μl 20% heptafluorobutyric acid, incubated at room temperature for 5 min and clarified by 5-min centrifugation at 16000 g. Peptides from clarified samples were desalted using C18 spin tips (Thermo Scientific) according to manufacturer’s instructions. Desalted peptides were dried under vacuum and redissolved in 0.1% formic acid prior to LC–MS analysis. Peptide concentration was measured at 205 nm on Nanodrop One (Thermo Scientific).

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